Recently, the development of plant biotechnology makes it possible to produce a transgenic plant. The technology for producing the transgenic plant can used in the two aspects. First aspect, it makes it possible to produce a valuable plant with new function which cannot be produced by conventional breeding technology. Second aspect, it makes it possible to use a plant as bioreactor. In other words, it makes it to produce valuable proteins and physiologically active substances derived from plant or animal using the transgenic plant or the transgenic plant cell line (Doran P M., 2000 Biochem. Engineering 11:199-204).
Many advantages of producing the useful proteins in the transgenic plant cell lines compared with the animal cell culture are as follows:
1) confirmation of security from infections of animal viruses
2) cheap culture cost in that the cost of plant cell culture can be reduced to 1/30 of animal cell culture's cost or ⅓ of microorganism culture's cost.
Thus, the studies for producing the useful proteins using transgenic cell lines as bioreactor have been increased rapidly. But the related study could be available practically only if the production protein of introduced gene reaches to a content of economic efficiency. The protein content produced in the transgenic plants or cell lines was only 0.001-0.4% out of total water-soluble protein contents excepting for Phytase (14.4%) and Xylanase (4.1%). Thus, most results of the studies are not available in the aspect of economic efficiency.
Another successful cases associated with the plant cell culture techniques are the production of secondary metabolites such as Shikonin (Japan Mitsui Petrochemical Industries Ltd.) and Taxol (Samyang Genex). But these cases have been limited to enhance the productivity of plant-originated substances. Recently as the genes related to these bioactive substances have begun to be found, the necessity of the development of molecular farming to enhance the expression of genes related to pathway of these bioactive substances has been noticed (Goossen et al., 2003 PNAS 100:8595-8600). Recently the gene related to pathway of the saponin biosynthesis, the medicinal component of ginseng have been found (Han et al., 2006 Plant Cell Physiol. 47:1653-1662; Tansakul et al., 2006 FEBS 580:5143-5149), thus there has been a need for generation of ginseng cultured roots with high saponin content using the metabolic engineering.
Consequently, the studies for promoters regulating the expression levels of foreign genes play a key role in the mass-production of the useful proteins originated from plant or animal and in high level expression of genes related to pathway for secondary metabolites using the plant tissue-culture.
Thus in order to develop these promoters, the important aspects to be considered are as follows:
1) the expression levels of foreign genes inserted into transformants are to be regulated. The genes for transformation can be used diversely according to the target characters. Therefore the expression levels of the genes are to be required variously. In order to accumulate the secondary metabolites in cultured cells, it is advantageous to induce high level expression of genes related to the biosynthesis pathway for the production of the physiological active substances. In order to produce useful proteins in a large quantities, it is required to develop the high-expressing promoter in the logarithmic growth phase, whereas in order to accumulate secondary metabolites in a large quantities, it is required to develop the high-expressing promoter in the stationary phase. Consequently, in order to regulate the expression levels of foreign genes minutely, promoters having diverse expression levels should be developed.
2) The promoters for transformation are to be protected by intellectual properties. At present the universal promoter, CaMV 35S promoter (Patent No. JP19931172-A1) have been patented. Therefore, if the useful substances are produced in a large quantities using the transformant lines containing the CaMV 35S promoter, the royalty have to be paid and the profitability decreases rapidly.
3) It is necessary to develop the suitable promoters for versatile tissue cultured cell lines. Tobacco BY-2 cell (Nicotiana tobacum L. cv.) has comparatively rapid growth rate and is the most suitable cell line for production recombinant proteins. But it was also reported that the medicinal proteins could be produced in the suspension cells of the soybean, tomato, and rice, and the hairy roots of tobacco.
For example, the productivity of human granulocyte-macrophage colony stimulating factor (hGM-CSF) using inducible promoter (amylase expression system) in rice cell lines was much higher than using the constitutive promoter, CaMV 35S in the tobacco cell lines (Shin et al., 2003 Biotech. and Bioengineering 82:778-783). In case that medicinal components exist in the roots such as ginseng, the promoter expressing highly in the cultured roots is needed. Thus in order to produce the useful proteins it is required to develop cell lines and promoters suitable for them respectively on a case-by-case basis.
Most of the root crops such as ginseng, Chinese bellflower and Lance Asiabell (Codonopsis lanceolata) are edible and have good pharmacological effects to be valuable crops. However, most of root crops are perennial (4˜6 years) and have long growth periods. The worse is difficult to extract DNA and RNA of root crops because they contain a lot of polysaccharides. It is also difficult to monitor the growth of storage roots growing up in the underground. Thus molecular breeding technology for the root crops has hardly been studied.
However, as genes related to bioactive substances have been begun to be reported recently, it have been required to develop the expression system for enhancement of the genes expression related to these biosynthesis pathway and accumulation bioactive substances in a large quantities.
Medicinal root crops comparatively require long cultivation periods so that it needs long time and high cost for producing and manufacturing economically.
Therefore, it is very important to develop the technology of molecular farming in the basis of study for promoters which can accumulate useful bioactive substances (for example saponin) in a short period and in a large quantities using the cultured roots and cultured cells.
Consequently, for the mass-producing useful foreign proteins and bioactive substances in an efficient and economic way using the plant tissue culture, the promoters directing high level expression of a foreign gene in tissue cultured cells such as cultured roots should be studied.
Meanwhile, it have been reported that Ran (Ras-related nuclear protein) GTPase is a protein present abundantly at meristematic tissues such as embryos in a developmental stage and suspension cultured cells. It plays a major role in regulation of cell growth and cell proliferation, and is essential for the translocation of RNA and proteins through the nuclear pores complex. In animal, Ran GTPase plays a key role in regulating nuclear processes through cell mitosis together with Ran-binding protein (RanBPs), RCC1 (a GEF, RanGEF) and RanGAP (a GAP) regulating these proteins.
Four kinds of Ran GTPase (AtRAN1, AtRAN2, AtRAN3, AtRAN4) derived from Arabidopsis have been identified through comparing the similarity of nucleotide sequences. There was a note that AtRAN4 of them could be induced by salt stress, but related result has not been reported yet. In addition, cDNA of Ran GTPase expressed highly in the roots of soybeans has been isolated. Ran GTPase cDNA has been identified from all tissues of tomatoes such as leaves (cotyledon, young leaves and senescencing leaves) of versatile stages of development, as well as roots and fruits. It has been found that Ran GTPase cDNA of tomato is a kind of nucleoprotein and suppresses the pim1 mutation of S. pombe (Robert et al., 1994 91:5863-5867).
But it has not been found how these various proteins translocate into the target places after synthesized or by what mechanism they are expressed in special tissues. The promoter of Ran GTPase gene in plants has been hardly studied.